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Abstract

Metabarcoding is an emerging genetic tool to rapidly assess biodiversity in ecosystems. It involves high-throughput sequencing of a standard gene from an environmental sample and comparison to a reference database. However, no consensus has emerged regarding laboratory pipelines to screen species diversity and infer species abundances from environmental samples. In particular, the effect of primer bias and the detection limit for specimens with a low biomass has not been systematically examined, when processing samples in bulk. We developed and tested a DNA metabarcoding protocol that utilises the standard cytochrome c oxidase subunit I (COI) barcoding fragment to detect freshwater macroinvertebrate taxa. DNA was extracted in bulk, amplified in a single PCR step, and purified, and the libraries were directly sequenced in two independent MiSeq runs (300-bp paired-end reads). Specifically, we assessed the influence of specimen biomass on sequence read abundance by sequencing 31 specimens of a stonefly species with known haplotypes spanning three orders of magnitude in biomass (experiment I). Then, we tested the recovery of 52 different freshwater invertebrate taxa of similar biomass using the same standard barcoding primers (experiment II). Each experiment was replicated ten times to maximise statistical power. The results of both experiments were consistent across replicates. We found a distinct positive correlation between species biomass and resulting numbers of MiSeq reads. Furthermore, we reliably recovered 83% of the 52 taxa used to test primer bias. However, sequence abundance varied by four orders of magnitudes between taxa despite the use of similar amounts of biomass. Our metabarcoding approach yielded reliable results for high-throughput assessments. However, the results indicated that primer efficiency is highly species-specific, which would prevent straightforward assessments of species abundance and biomass in a sample. Thus, PCR-based metabarcoding assessments of biodiversity should rely on presence-absence metrics.

Author Comment

This is a revised version of the manuscript which was submitted to PLOS ONE in January 2015. It is currently in the second round of peer review. A short YouTube video summarising the findings of this paper is available: https://www.youtube.com/watch?v=VifqvI5JeDM

S9 Figure. Experiment II: OTUs assigned to taxa

Additional Information

Competing Interests

The authors have declared that no competing interests exist.

Author Contributions

Vasco Elbrecht conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote the paper, prepared figures and/or tables, reviewed drafts of the paper.

Florian Leese conceived and designed the experiments, contributed reagents/materials/analysis tools, wrote the paper, reviewed drafts of the paper.

DNA Deposition

The following information was supplied regarding the deposition of DNA sequences:

The Illumina sequencing data are available via the Short Read Archive (accession numbers SRS731403 and SRS733820 ).

Funding

This work was supported by a grant of the Kurt Eberhard Bode Foundation to FL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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May I congratulate you on the results of this eloquent and much needed experiment - well done and clearly a lot of work! The R squared data that you show reflecting biomass and normalised number of reads is amongst some of the most convincing I have seen and certainly worth future citations. Nevertheless, unless you have specifically tested priming efficiency, the differences in number of reads per taxon may also be due to different mitochondrial densities. Similar results have been shown by Bruce Deagle and colleagues in Molecular Ecology Resources from their diet study a couple of years ago. Whether or not these are artefacts of primer binding, or different densities of mitochondria remains to be seen (assuming effective DNA extraction etc.)? Nevertheless, I think that reducing metabarcoding data to presence and absence on the basis of your results may be throwing the baby out with the bathwater (as they say in English), in that your results suggest that you can track abundances of single taxa in space and time, but not compare the relative abundance of one taxon with another as would be predicted according to both primer bias and mitochondrial concentrations.

Hope that this helps and good luck with the rest of your journey to get this out to publication.

Thank you very much for your feedback. I agree different densities of mitochondria can certainly influence the read abundance to some degree (also, in this study the amount of exoskeleton might to a low degree, when measuring the weight of our specimens). However, I doubt that variability in mitochondrial densities will lead to differences of several magnitudes, thus I would account the majority of the effects we can see to primer bias. However, I appreciate your suggestion and we will make sure to make clear that variability in mitochondrial densities can additionally bias abundance data, in future publications! This paper has already been accepted for publication in plos one, and will be online soon, so I'm afraid we can't add this aspect to the current manuscript.

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